The accurate and efficient replication of genomic DNA is critical for all organisms. Errors in this process can lead to mutations and ultimately to cancer. The important task of DNA replication is performed by a multi- protein complex, the replisome, which shares broadly conserved features across all domains of life. The central component of the replisome is DNA polymerase, the enzyme that synthesizes DNA with the help of processivity factors and other accessory proteins. In eukaryotes, three different polymerases are needed for chromosomal replication. This is in stark contrast to the model bacterial system, Escherichia coli, which has a single replicative polymerase. More recently, it has been discovered that two replicative polymerases, PolC and DnaE, are required in low-GC Gram-positive bacteria like Bacillus subtilis. Questions remain about the role of these polymerases and the mechanisms by which their activity is coordinated. Unlike PolC, DnaE lacks a proofreading domain and is error-prone; thus the proper regulation of DnaE activity during replication is critical for the cell. A detailed molecular-level understanding of how polymerase activity is coordinated by B. subtilis will provide insights into how replicative polymerases are regulated in more complex eukaryotic systems. This research will utilize novel single-molecule approaches to address the following specific aims: Aim 1: Determine the architecture and organizing principles of the B. subtilis replisome. It is becoming clear that the organization of the replisome in B. subtilis differs in key ways from that in E. coli, likely becaue of the need to coordinate two different polymerases. This aim will utilize in vivo single-molecule imaging of fluorescent fusion proteins to determine the copy number of important replisome components like the polymerases PolC and DnaE, the sliding clamp DnaN, and the ? subunit of the clamp loader complex. The protein-protein interactions that help to organize the replisome will be identified by making targeted mutations to domains implicated in these interactions. Aim 2: Investigate how PolC and DnaE activity is coordinated at the replication fork. In a current model for the role of PolC and DnaE, the two polymerases act sequentially on the lagging strand, meaning that polymerase exchange must occur repeatedly during replication. How such polymerase switching events are regulated and how interactions with replisome components mediate the exchange is unknown. These questions will be addressed by utilizing single-molecule imaging to measure the lifetimes of PolC and DnaE at the replication fork in live B. subtilis cells. The dynamics of individual PolC and DnaE molecules, which are obscured in ensemble biochemical experiments, will help to elucidate their roles during replication. A more targeted mechanistic investigation of polymerase exchange will be performed using an in vitro single- molecule DNA synthesis assay involving a minimally reconstituted replisome. This assay will reveal the kinetics of polymerase exchange and will help identify the protein-protein interactions involved in this process.